Salivary secretions maintain the health of the oral cavity. Building on our past studies of saliva formation and its alteration during pathology, we are developing novel approaches to treat salivary gland dysfunction primarily using principles of gene therapy and tissue engineering. During this reporting period we have made considerable progress in many facets of our work. Perhaps most important are those studies focused on fundamental questions necessary to move gene therapy into the clinic for phase I trials. Three key questions were addressed, and the following answers were obtained. First, what is the best recombinant vector to use for stable transgene expression in salivary glands? At present the answer appears clearly recombinant adenoassociated virus serotype 2 (rAAV2). Using a rAAV2 vector encoding human erythropoietin (hEpo) we have shown the secretion of functional hEpo into the bloodstream for 48 weeks in mice. Second, can transgene expression in salivary glands be regulated? Yes, as during this reporting period we have shown that it is possible to regulate transgene expression using the rapamycin-based dimerizer drug system. Employing two adenoviral vectors to deliver the components of this transcription regulation system, we showed that rapamycin is able to function as a control switch for transgene expression in rat salivary glands. Importantly, in the absence of rapamycin, no transgene expression is detected. Third, can results obtained in rodent model experiments be scaled to larger animals? Yes, and we have recently shown that using a comparable multiplicity of infection (dose; adenoviral infectious units/ml infusate), we are able to obtain similar levels of transgene expression in mouse and minipig salivary glands. Our translational research studies are directed at two major causes of salivary hypofunction; Sjogren?s syndrome and damage from ionizing radiation received during therapy for head and neck cancers. As we previously reported, female non-obese diabetic (NOD) mice develop spontaneous autoimmune sialadenitis and loss of salivary flow, and are an excellent model of Sjogren?s syndrome. We constructed a rAAV2 vector encoding human IL-10, (rAAVhIL10) and delivered this vector (or a control) by retrograde ductal instillation to submandibular glands of mice either before or after the onset of sialadenitis. After administration of rAAVhIL10, salivary flow rates were significantly higher, and inflammatory infiltrates significantly reduced, from results seen with control vector-treated mice. Additionally, treated mice exhibited significantly lower blood glucose, and higher serum insulin, levels. These studies show that expression of hIL-10 directed by a rAAV2 vector can have disease-modifying effects in salivary glands of NOD mice. The second translational research goal is to protect salivary glands from, or repair salivary glands after, radiation damage. During this reporting period, we have explored both gene transfer and novel pharmacological approaches to achieve these goals. Recently, we achieved a significant and exciting advance with the pharmacological approach. We tested the ability of the stable free nitroxide radical tempol to minimize salivary gland irradiation damage. In conjunction with mouth/nose shielding, a single intraperitoneal injection of tempol 10 min prior to irradiation completely prevented radiation (15 Gy)- induced salivary hypofunction in C3H female mice, i.e., salivary flow rates were equivalent to those of non-irradiated, tempol-treated mice. We also have extended studies initially reported last year examining whether bone marrow progenitor cells are capable of transdifferentiating into buccal mucosal and/or salivary epithelial cells. Four to six years after male-to-female marrow cell transplantation, all five female recipients studied by us exhibited Y-chromosome positive buccal cells (0?8% to 12?7%) that were cytokeratin 13 positive. In > 9700 cells examined, we detected only one XXXY positive cell (0?01%) and one XXY cell (0?01%), both of which may have arisen when a XY-cell fused with a XX cell. Thus, it appears that male bone marrow cells migrate into the cheek and differentiate into epithelial elements, a phenomenon that does not depend on cell fusion. We are continuing to investigate whether this same phenomenon occurs in salivary glands.